Antithrombogenic article containing lysozyme and heparin adsorbed on a substrate

ABSTRACT

An article adapted for applications where there is contact with blood and especially in medical applications is prepared by adsorbing the enzyme, lysozyme or derivative thereof, onto a substrate and then adsorbing heparin or a heparin-based material to the enzyme. The substrate is preferably a metal or polymeric material.

TECHNICAL FIELD

The present invention relates to the field of heparinization ofmaterials for the purpose of imparting thereto a heparin layer whichinhibits in contact with blood the adhesion of thrombocytes and theadsorption of blood proteins. Thus, the article according to theinvention is especially suited for applications where there is contactwith blood, e.g. medical applications. It is true that the methodologyof heparinizing a material for the above-mentioned purpose is previouslyknown per se, but the present invention relates to a novel, alternativemethod of adhering the heparin to the substrate, via a special, noveltype of a pre-adsorbed layer. In addition to the above-mentioned articlethe invention relates to a process for the preparation thereof as wellas to the use of said article for medical applications.

BACKGROUND OF THE INVENTION

To accomplish blood compatibility for different materials in contactwith blood one of the most important methods has been to heparinize thesurface thereof. Thus, the heparin layer on the surface inhibits, as wasmentioned above, the adhesion of thrombocytes and the adsorption ofblood proteins. Furthermore, the heparin must be enzymatically active inthe blood coagulation process, which calls for specific necessities asto molecular conformation and mobility relative to the surface.

Two main principles for the heparinization have previously beenutilized. The first one is based on colloidal precipitation, e.g.through complex-formation between amphiphilic amines and heparin. Thesecond one utilizes the possibility of covalently bonding the heparin tothe surface. However, these known principles have some limitations whichmeans that there is continuous research for alternative or improvedmethods for the heparinization.

SUMMARY OF THE INVENTION

The present invention relates to an alternative or improved techniquefor the heparinization of surfaces, which technique eliminates or atleast reduces the limitations of the prior art while at the same timeimparting thereto, at least for certain applications, additionaladvantageous properties which have not been obtainable by the previouslyutilized technique. More specifically we have found that a specificprotein, viz. lysozyme, possesses unexpected affinity for heparin andgives an outstanding adhesion to different substrate surfaces. Theunexpectedly good results which have been obtained by the proteinaccording to the invention will be described more in detail below, butprimarily it can be mentioned that a very good adhesion to metalsurfaces has been obtained, which is material in which previously knownmethods have shown deficiencies.

Lysozyme is a protein that is present in low concentrations in blood.Already there is an interesting advantage since the invention is basedon the utilization of a substance which is non-foreign to the humanorganism. In addition thereto another interesting property of lysozymeis its antimicrobial properties which impart to the novel heparinizedsurface an antimicrobial activity; a security factor in storage andhandling.

More specifically the article according to the invention ischaracterized in that heparin or a heparin-based material is adhered orbonded to the substrate via a layer of lysozyme or a derivative thereofwhich is pre-adsorbed to said substrate.

As was indicated above the novel technique according to the inventionhas been shown to work especially well for metal surfaces, in whichpreviously known heparinizing methods have shown limitations. However,the invention is also applicable to other substrates which are chosenper se in accordance with previously known techniques, i.e. primarilysuch substrates that have has previously been desired to heparinize forthe purpose of imparting to the same improved properties in applicationswhere there is contact with blood. Examples of such materials arepolymeric materials and glass. As far as polymeric materials areconcerned it should be noted that the present invention is especiallyinteresting in connection with polymeric materials of the so called lowenergy type. Low energy type means polymeric materials that are notwetted by water but by organic solvents.

Concerning the term "lysozyme or derivatives thereof" it should beunderstood that of course the invention is not limited to the use oflysozyme per se, but it is also possible to choose any derivativethereof which gives the corresponding or similar properties. Such achoice may for instance depend on better solubility in the desiredsolvent for a derivative than for lysozyme per se. Examples ofutilizable derivatives may include salts, such as the chloride salt.Moreover, the invention is of course intended to cover such cases wherethe lysozyme has been modified within the molecule at a position or sitethat does not have any direct connection with the effect of theinvention, i.e., a modification that does not change the desiredproperties according to the present invention.

The same requisite concerning heparin is that heparin per se does nothave to be utilized to obtain the desired effect. Thus, the expression"heparin-based material" is intended to cover those heparin compoundswhich give a corresponding or similar effect, reference in this contextbeing made to the prior art which discloses numerous examples of heparincompounds for the purpose referred to. Thus, in this connection theinvention does not differ from the prior art.

Those applications for which the article claimed is especially wellsuited are also selected in accordance with the prior art, and thus neednot be described herein. However, certain improvements of the propertiesor additional advantageous properties are obtained by the presentinvention, medicinal uses or other applications will become even moreapparent in the present invention than described in the prior art.

The process according to the invention is characterized by firstcontacting the substrate with a solution of lysozyme or the derivativethereof to form a lysozyme layer and then exposing the substrate withits lysozyme layer to a heparin or heparin-based solution to adhere orbond the heparin or the heparin-based material to said lysozyme layer.

As is often the case for surfaces which are to be coated, such surfaceshave to be comparatively clean to obtain the desired result. This istrue also in the present invention, especially in the case where thesubstrate is a metal. In such a case the surface should be very clean,and is comprised of metal or metal oxides. In the ideal case this meansthat the surface should be cleaned or purified in a so called plasmacleaner and immediately thereafter transferred into distilled water.Alternatively, a consecutive washing in lye, acid and distilled water isacceptable. For a plastic surface, especially a low energetic one, thematerial is cleaned in water with a detergent and then an organicsolvent. Concerning other substrates in principle those cleaning methodswhich have previously been utilized in connection therewith areapplicable.

After said cleaning of the substrate surface, if required, the substrateis contacted with the lysozyme solution, which is commonly a watersolution or an aqueous solution. Distilled water is often preferredrelative to a buffer solution. In order to obtain a lysozyme layer thesolution should have a concentration of at least 0.1 percent by weight.The upper limit is not especially critical to accomplish the desiredeffect, but generally the concentration should not exceed 10 percent byweight, since otherwise viscosity effects will interfere with theprocess. An especially preferable range for the concentration oflysozyme or derivative thereof is 0.1-2 percent by weight.

The residence time of said stage of treatment should be at least 15minutes, preferably about 20 minutes. Such a period is normally requiredto attain a plateau value for the adsorption of lysozyme. Once saidplateau or maximum value has been attained there is normally no reasonto further extend the residence time, which generally means that saidresidence or treatment time is within the range of 15-30 minutes.

After said treatment with the lysozyme solution the substrate should berapidly rinsed in water and then directly exposed to a heparin solutionor a heparin based solution. Thus, it has been discovered, especially inconnection with metals, that drying should not or must not be performedbetween the two coating stages, in order to obtain the optimum effect.

Also the solution of heparin or heparin-based compound is preferablywater-based. For adsorption reasons the heparin solution concentrationshould be above 0.05 percent by weight, especially above 0.1 percent byweight. In this case the upper limit is not especially critical, and anyadditional effect is negligible at a concentration value exceeding about5 percent by weight. Therefore, a generic range is 0.05-5, especially0.1-5, percent by weight. However, in many cases said concentrationshould not even exceed about 2 percent by weight, since the viscositywill cause interferences. Thus, the specially preferred range is 0.1-2percent by weight. However, concerning the heparin treatment inprinciple all applications from the prior art can be utilized, i.e. saidstage is principally performed per se in accordance with the guide-linesof the prior art in this field.

The exposure time of the heparin solution or the heparin based solutionis generally at least 20 minutes, preferably about 30 minutes, and morepreferably between 20-45 minutes.

After exposure to the heparin solution the substrate is rinsed indistilled water, whereupon it is allowed to dry or is dryed afterdraining the excess solution. By rinsing in distilled water beforedrying the amount of heparin can be reduced to a monomolecular layer.However, for most applications a certain surface excess of dissolvedadsorbed heparin is preferred.

Both of the above-mentioned surface treatments are preferably performedat room temperature. A slightly bigger temperature can be utilized ifdesired, but generally the temperature should not exceed about 50° C.,since structural changes may appear in the lysozyme.

Finally the invention relates to the use of the above-defined article orof an article prepared by the process defined above, for medicalapplications where there is contact with blood. It should be noted thatof course the term "medical applications" should be interpreted in abroad meaning; the use is not specifically limited to therapeuticaltreatments only.

The invention will now be further described by means of the followingnon-limiting examples. The percentages used therein relate topercentages by weight unless otherwise specifically stated.

EXAMPLE 1

A commercially available lysozyme from poultry egg white was checked bygel electrophoresis to be free of other egg white proteins. The lysozymewas then de-salted by dialysis. A solution of 0.5 percent by weight oflysozyme in distilled water was then prepared. Metal cannulae weresubmersed in a bath of said solution for 20 minutes. Said metal cannulaewere pre-cleaned for 5 minutes in a so called plasma cleaner at an airpressure of 5 torr. They were taken from the bath, rinsed with distilledwater and immediately transferred to a bath consisting of a 0.1% heparinsolution in distilled water. After 30 minutes the cannulae were removedfrom the bath, rapidly rinsed with distilled water and allowed to dry ina sterile chamber at 30° C. In this example metal cannulae having aheparin coating adhered via a pre-adsorbed layer of lysozyme wereobtained.

EXAMPLE 2

Catheters of polyethylene were washed in a one percent Triton X-100solution and then in ethanol (96%). Said catheters were submersed in a0.1% lysozyme solution in distilled water. After about 20 minutes theywere placed in a bath of distilled water, whereupon they were furthertransferred to a 0.1% heparin solution in distilled water. After 30minutes the catheters were washed and were then allowed to dry thusforming articles of the present invention.

CLINICAL INVESTIGATION OF HEPARINIZED STEEL TUBES ACCORDING TO THEINVENTION

Several methods have been utilized in order to determine thrombogenicityfor artificial materials. A previously utilized method is one in whichsteel tubes are inserted into blood vessels and are incubated in thevessel. During incubation the coagulation system reacts by adsorbingproteins on the extraneous surface and adhesion of thrombocytes andpossible thrombosis of the inserted tube occurs. From an experimentalpoint of view said methodology is a good method to study the formationof thrombosis in arteries as well as in veins.

Lately methods which utilize labeled radisisotopes have been used forstudies of thrombogenicity. However, to estimate the thrombogenicity ofsteel tubes the previously used technique with an intravascularinsertion of the steel tube and determination of the weight differencesbefore and after incubation is the best method for an optimumdetermination of the thrombogenicity of the material. Steel tubes havinga diameter of 4 mm, a length of 25 mm and a thickness of 0.1 mm wereheparinized in accordance with Example 1 above.

MATERIALS AND METHODS

Three sheep, weighing about 40 kg. were anesthetized with pentobarbitalat an initial concentration continuously in fused at a rate of 30 mg/kg.Afterwards the pentobarbital was 7.5 mg/min. The sheep were intubatedand respirator ventilated with 40% O₂. The respirator frequency was20/min, volume 10 1/min. Both carotids were explored and opened by asmall longitudinal incision and the 25 mm long steel tube, tapered andpolished, was inserted. In carotid on one side there was inserted aheparinized tube and in the other side a non-heparinized tube. Betweendifferent incubation periods the sides were changed. After preliminarytesting the incubation time was selected as being 15 minutes.

RESULTS

25 incubation periods were performed. In all these the thrombus weightswere considerably less on the heparinized tube than on thenon-heparinized one (32+4 mg as compared to 210+10 mg). In additionthere were additional thrombus masses in the vessel in seven cases whenthe steel tube was removed. All these thrombus masses were innon-heparinized tubes and had weights of 96, 201, 143, 369, 374, 216 and199 mg.

Statistical calculations when using the student's paired t-test gave at-value of t=9.20, df 25, reflecting a considerably significantreduction of thrombogenicity in the heparinized tubes.

We claim:
 1. An antithrombogenic article consisting essentially of asubstrate, an enzyme selected from the group consisting of lysozyme, alysozyme derivative and a mixture thereof, which enzyme is preadsorbeddirectly to said substrate to form an enzyme layer, and anantithrombotic compound, selected from the group consisting of heparin,a heparin-based material and a mixture thereof, said antithromboticcompound being adhered to said substrate by adsorption to said enzymelayer.
 2. An article according to claim 1, wherein the substrate is ametal.
 3. An article according to claim 1, wherein the substrate is apolymeric material which is not wetted by water but by an organicsolvent.
 4. An article according to claim 1, wherein said antithromboticcompound is heparin.
 5. An article according to claim 1, wherein saidantithrombotic compound is a heparin-based compound.
 6. An articleaccording to claim 1, wherein said enzyme is lysozyme.
 7. An articleaccording to claim 1, wherein said enzyme is a lysozyme derivative.
 8. Aprocess for the preparation of an antithrombogenic article, said processconsisting essentially of contacting a cleaned substrate with a solutioncomprising an enzyme selected from the group consisting of lysozyme, alysozyme derivative and a mixture thereof, which enzyme is preadsorbeddirectly to said substrate to form an enzyme layer and exposing thesubstrate containing the enzyme layer to a solution containing anantithrombotic compound selected from the group consisting of heparin, aheparin-based material and a mixture thereof, to adsorb saidantithrombotic compound to the enzyme layer.
 9. A process according toclaim 8, further comprising the step of rinsing with distilled waterafter forming the enzyme layer.
 10. A process according to claim 8,wherein said solution containing an antithrombotic compound is anaqueous solution.
 11. A process according to claim 8, wherein theconcentration of enzyme in solution is between 0.1 to 10 percent byweight.
 12. A process according to claim 11, wherein the concentrationof enzyme in solution is between 0.1 to 2 percent by weight.
 13. Aprocess according to claim 8, wherein the concentration of saidantithrombotic compound in solution is between 0.05 to 5.0 percent byweight.
 14. A process according to claim 13, wherein the concentrationof said antithrombotic compound in solution is between 0.1 to 2 percentby weight.
 15. A process according to claim 8, wherein said enzymesolution is contacted with the substrate for at least 15 minutes.
 16. Aprocess according to claim 15, wherein said enzyme solution is contactedwith the substrate for a period between 15 to 30 minutes.
 17. A processaccording to claim 8, wherein the exposure to the antithromboticcompound solution is for a period of at least 20 minutes.
 18. A processaccording to claim 17, wherein the exposure to the antithromboticcompound solution is for a period between 20 to 45 minutes.
 19. Aprocess according to claim 8, wherein said enzyme layer remains wetbefore exposure to said antithrombotic compound solution.
 20. A processaccording to claim 13, wherein said enzyme solution is contacted withthe substrate for at least 15 minutes.
 21. A process according to claim20, wherein said enzyme solution is contacted with the substrate for aperiod between 15 to 30 minutes.
 22. A process according to claim 11,wherein the exposure to the antithrombotic compound solution is for aperiod of at least 20 minutes.
 23. A process according to claim 22,wherein the exposure to the antithrombotic compound solution is for aperiod between 20 to 45 minutes.
 24. A process according to claim 13,wherein the exposure to the antithrombotic compound solution is for aperiod of at least 20 minutes.
 25. A process according to claim 24,wherein the exposure to the antithrombotic compound solution is for aperiod between 20 to 45 minutes.
 26. A process according to claim 15,wherein the exposure to the antithrombotic compound solution is for aperiod of at least 20 minutes.
 27. A process according to claim 26,wherein the exposure to the antithrombotic compound solution is for aperiod between 20 to 45 minutes.
 28. A process according to claim 11,wherein said enzyme layer remains wet before exposure to saidantithrombotic compound solution.
 29. A process according to claim 13,wherein said enzyme layer remains wet before exposure to saidantithrombotic compound solution.
 30. A process according to claim 15,wherein said enzyme layer remains wet before exposure to saidantithrombotic compound solution.
 31. A process according to claim 17,wherein said enzyme layer remains wet before exposure to saidantithrombotic compound solution.
 32. A process for the preparation ofan antithrombogenic article said process consisting essentially of firstcontacting a cleaned metal substrate with a solution containing anenzyme selected from the group consisting of lysozyme, a lysozymederivative and a mixture thereof, which enzyme is preadsorbed directlyto said substrate to form an enzyme layer and exposing the substratewith its enzyme layer to a solution containing an antithromboticcompound selected from the group consisting of heparin, a heparin-basedmaterial and a mixture thereof, to adsorb the antithrombotic compound tothe enzyme layer.
 33. A process according to claim 32, furthercomprising the step of rinsing the enzyme layer with distilled waterprior to exposing the enzyme layer to the solution containing theantithrombotic compound.
 34. A process according to claim 32, whereinsaid solution containing an antithrombotic compound is an aqueoussolution.
 35. A process according to claim 32, wherein said enzymesolution is contacted with the substrate for at least 15 minutes.
 36. Aprocess according to claim 35, wherein said enzyme solution is contactedwith the substrate for a period between 15 to 30 minutes.
 37. A processfor the preparation of an article said process consisting essentially offirst contacting a cleaned polymeric substrate with a solutioncontaining an enzyme selected from the group consisting of lysozyme, alysozyme derivative and a mixture thereof, which enzyme is preadsorbeddirectly to said substrate to form an enzyme layer and exposing thesubstrate with its enzyme layer to a solution containing anantithrombotic compound selected from the group consisting of heparin, aheparin-based material and a mixture thereof, to adsorb theantithrombotic compound to the enzyme layer.
 38. A process according toclaim 37, further comprising the step of rinsing the enzyme layer withdistilled water prior to exposing the layer to the solution containingthe antithrombotic compound.
 39. A process according to claim 37,wherein said solution containing the antithrombotic compound is anaqueous solution.
 40. A process according to claim 37, wherein theconcentration of said antithrombotic compound in solution is between0.05 to 5.0 percent by weight.
 41. A process according to claim 40,wherein the concentration of said antithrombotic compound in solution isbetween 0.1 to 2 percent by weight.